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Worm PCR Protocol
Single Worm PCR Protocol What you will need: 1. PCR strips 2. Thermocycler 3. Worms 4. Worm Lysis Buffer + Proteinase K 5. Primers for YFG (100mM) 6. 10x Single-worm PCR Buffer 7. Taq 8. MgCl2 (50mM) 9. dNTP mix (10mM) 10. ddH2O 11. Restriction Enzyme and buffer (if RFLP is necessary) Making the things you will need Worm Lysis Buffer (WLB) Combine the Following in a 15ml conical tube. * ddH2O 8980µl * Tris pH 8.2, 1M 100µl * KCl, 1M 500µl * MgCl2, 1M 25µl * Tween 20, 100% 45µl * Gelatin, 2% Stock 250µl Total=9.9ml * Steriflip the mixture and aliquot 990µl per micro centrifuge tube and store at -20°C. Worm Lysis Buffer + Proteinase K (WLB+PK) * Take 1 990µl WLB tube and add 10µl Proteinase K (6ml/ml). * Re-label the tube and store at -20°C 10X Single Worm PCR Buffer Combine the Following in a micro centrifuge tube. * ddH2O 383µl * Tris pH 8.3, 1M 100µl * KCl, 1M 500µl * MgCl2, 1M 12µl * Gelatin, 2% Stock 5µl Total=1ml * Steriflip the mixture and store at -20°C. If you want, you can make a larger batch and just aliquot the samples to micro centrifuge tubes. Lysing the Worm # In a labeled PCR strip, add 10 µl of WLB+PK per well. To each well, add a single worm. # Spin briefly to ensure that the worm and WLB+PK are at the bottom of the tube. # Flash freeze the worms in Liquid nitrogen for 10 minutes. Allow the worms to thaw to room temperature before lysing the worms in the Thermocycler. (The worms may be stored indefinitely at -80°C) # Incubate the reaction in the thermocycler using the protocol called "wormlysis" # After being lysed, the worms may be stored at -20°C for a short period of time or at -80°C indefinitely. PCR 1. Prepare your Master Mix with the appropriate primers by following the chart below. An excel version of the cart can be found in the dropbox under worm protocols. 2. When the Master Mix is prepared, aliquot the appropriate amount into the wells of PCR strips. 3. Add 2.5µl of the Worm Lysate to the PCR strip wells. 4. LABEL LABEL LABEL LABEL!!! 5. Run the protocol “Worm PCR”- you may need to change the annealing temperature depending on the primers you are using. You may also need to change the extension time depending on the length of your desired product (1min per 1Kbp). 6. After the PCR has finished, run the samples on an agarose gel. If you need to RFLP DO NOT add loading dye directly to the PCR samples. Instead, get a sheet of parafilm, and put 2µl dots of loading dye spaced out on the sheet. Then take 6µl of the PCR sample, mix them on the sheet and then load that into the gel. = Restriction Fragment Length Polymorphism-RFLP = If the gene you are trying to check has only a point mutation instead of a deletion, you will need to use a process called RFLP. 1. Using a sequence mapping software (Ugene) and WormBase, determine exactly where the mutation is in both the WildType and Deletion. 2. Using NEB Cutter, enter both sequences around the mutation site (about 8-10bp on either side should be enough). NEB cutter will give you a series of restriction sites for both sequences. You want to find a restriction site that is one sequence but not the other. This will allow you to digest the PCR products giving you one, two or three bands depending on the genotype. 3. If we do not have the restriction enzymes, you will have to order them (there are multiple boxes in the labs -20°C freezers) 4. Follow the directions on the provided card, or look up the protocol online for your particular enzyme. 5. After the digest is complete, run your samples on an agarose gel. KEEP YOUR ENZYMES ON ICE ALWAYS!!!!